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Center for Microscopy and Image Anaylsis
Introduction to light microscopy
Imaging with light / Overview of techniques
Urs Ziegler
Light interacting with matter
Absorbtion
Refraction
Diffraction
Scattering
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Light interacting with matter
Absorbtion
Refraction
Diffraction
Scattering
Light interacting with matter
Light emitted from fluorochromes
How is an image formed?
Why are there limits in resolution?
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Introduction to light microscopy
Imaging with light / Overview of techniques
Image formation in a nutshell
Resolution limits
Light emission from molecules and fluorescent imaging
Methods and techniques in microscopy
Widefield microscopy
Confocal laser scanning microscopy
Fluorescence energy transfer
Fluorescence recovery after photobleaching
In vivo microscopy
Selective plane illumination microscopy
Superresolution techniques
Correlative techniques – light and electron microscopy
Fundamental Setup of Light Microscopes
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Fluorescence in microscopy
DNA
Bax
Mitochondria
Cytochrome C
DNA
Bax
Mitochondria
Cytochrome C
DNA
Bax
Mitochondria
Cytochrome C
Fluorescence in microscopy
Advantages:
Very high contrast resulting in high sensitivity
Tagging of specific entities possible
Excitation / emission allows for various variants of microscopy techniques
Jablonski scheme
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Diffraction at an aperture or substrate
Disturbance of the electric field of a planar wave front by diffraction upon passage through an aperture
A mixture of particles diffracts an incident planar wave front inversely proportional to the size of particles
Resolution and aperture angle
Concept:
Object is approximated with self luminous points
Image of each individual point is not influenced by any other points
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Spatial resolution in x,yand z
Implications:
Objects smaller than the resolution limit of the chosen objective will always be 1Airy disk
Objects larger than the resolution limit of the chosen objective will always be the size of the object convolved with the optical transfer function
Note: the optical transfer function is a function describing how the imaging is occurring in the microscope
1 µm
Crossection
focal plane
0.1 µm bead
Reality
Theory
1 µm
Crossection
Resolution and size of Airy disk
Concept: an image of an extended object consists of a pattern of overlapping diffraction spots
Resolution: the larger the NA of the objective, the smaller the diffraction spots (airy disks).
Note: this theme of diffraction limited spots and their separation in space and time will again be used and taken up in superresolution microscopy.
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Resolution and Rayleigh criterion
Resolving power of microscope:
0.61 λConcept: an image of an extended object consists of a pattern of overlapping diffraction spots
Resolution: the larger the NA of the objective, the smaller the diffraction spots (airy disks).
a) Single diffraction pattern
b) Two Airy disks with maximum of one overlapping first minimum of the otherobjects just resolved
c) Two Airy disks with maximum of one overlapping the second minimumobjects well resolved
Spatial resolution in x,y and z
Objects are (always) 3 dimensional
The resulting ‘image’ will also be a 3D ‘image’ in the image space
Again:
an image of an extended object consists of a pattern of overlapping diffraction spots
1 µm
Crossection
focal plane
0.1 µm bead
Reality
Theory
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Resolution and size of Airy disk
Objects are (always) 3 dimensional
The resulting ‘image’ will also be a 3D ‘image’ in the image space
Again:
an image of an extended object consists of a pattern of overlapping diffraction spots
Take home:
In widefield microscopy the out of focus information is increasing the background and results in low contrast images
Resolution limits
0.61 λ λ
These formula are used for the calculation of resolution in widefield microscopy.
In other techniques like confocal laser scanning, multiphoton microscopy, etc other formula are used.
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Comparison of widefield and confocal microscopy
Confocal microscopy has a very high signal to noise ratio (prominent in thick samples)
Confocal microscopy allows well resolved 3D imaging (without any image processing)
Image acquired with a widefield microscope
Image acquired with a confocal microscope
λ
NAPHn
NAnnem 288.0dz
2
22
2
Confocal laser scanning microscopy
Sample is excited by a diffraction limited point of a focused laser spot
Emitted fluorescent light from focus is focused at pinhole and reaches detector
Emitted fluorescent light from out-of-focus is also out-of- focus at pinhole and largely excluded from detector
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Schätzle, P., J. Ster, D. Verbich, R.A. McKinney, U. Gerber, P. Sonderegger, and J.M. Mateos. 2011. Rapid and reversible formationof spine head filopodia in response to muscarinic receptor activation in CA1 pyramidal cells. The Journal of physiology. 589:4353-64.
Spinning disk microscopy
Increase acquisition speed
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Performance comparison between the high‐speed Yokogawa spinning disc confocal system and single‐point scanning confocal systems
Journal of MicroscopyVolume 218, Issue 2, pages 148-159, 27 APR 2005 DOI: 10.1111/j.1365-2818.2005.01473.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.1365-2818.2005.01473.x/full#f3
Fluorescence recovery after photobleaching
Image sample using widefield microscopy
Bleach defined region using intense illumination
Measure fluorescence intensity over time in the photobleachedregion
Time for recovery of fluorescence is an indication for:
Diffusion
Mobility
Binding
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Fluorescence recovery after photobleaching
Image sample using widefield microscopy
Bleach defined region using intense illumination
Measure fluorescence intensity over time in the photobleachedregion
Time for recovery of fluorescence is an indication for:
Diffusion
Mobility
Binding
Measuring properties: e.g. Ca2+
Measurement:ratio imaging with an excitation of340 and 380 nm
Walch, M., E. Eppler, C. Dumrese, H. Barman, P. Groscurth, and U. Ziegler. 2005. Uptake of granulysin via lipid rafts leads to lysis ofintracellular Listeria innocua. J Immunol. 174:4220-4227.
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Fluorescence resonanceenergy transfer
Palmer, A.E., and R.Y. Tsien. 2006. Measuring calcium signaling usinggenetically targetable fluorescent indicators. Nature protocols. 1:1057-65.
McCombs, J.E., and A.E. Palmer. 2008. Measuring calcium dynamicsin living cells with genetically encodable calcium indicators. Methods(San Diego, Calif.). 46:152-9.
Transfer of energy from donor to acceptor without light emission from donor.
Ratio imaging of donor / acceptor or measurment ofincrease in acceptor emission when exciting the donor.
Multiphoton microscopy
Imaging deep into tissue
Pulsed infrared laser (700-1500nm) excitesfluorochromes by multiphoton absorbtion
Excitation in a small volumedefined by the probability(densitiy of photons high) of a simultaneous multiphoton absorbtion
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Multiphoton microscopy
Imaging in scattering tissueand deep into tissue
Pulsed infrared laser (700-1500nm) excitesfluorochromes by multiphoton absorbtion
Excitation in a small volumedefined by the probability(densitiy of photons high) of a simultaneous multiphoton absorbtion
All fluorescent photonsprovide useful signals.
Helmchen and Denk, Nature Methods2005
Multiphoton microscopy
Helmchen, F., and W. Denk. 2005. Deep tissue two-photon microscopy. Nature methods. 2:932-40.
Living mouse: kidney (Hoechst, 10kD dextran FITC, 150kD dextran Texas Red
Brain
Kidney
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Selective Plane Illumination Microscopy
SPIM
4D imaging
Light-sheet-imaging technique
Better signal-to-noise ratio
Low phototoxicity
Selective Plane Illumination Microscopy
Keller, P.J., and E.H.K. Stelzer. 2008. Quantitative in vivo imaging ofentire embryos with Digital Scanned Laser Light Sheet FluorescenceMicroscopy. Current opinion in neurobiology. 18:624-32.
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Total internal reflection fluorescence microscopy
TIRF
Laser excitation light is directed at a tissue sample through a glass slide at a specific, oblique angle (critical angle)
Most of the light is reflected at the interface between glass and the tissue sample (total internal reflection)
Induction of an evanescent wave parallel to the slide
Decay of the evanescent wave over 200 nm
Stephens, D.J., and V.J. Allan. 2003. Light microscopy techniques for live cell imaging. Science (New York, N.Y.). 300:82-6.
Total internal reflection fluorescence microscopy
GFP- paxilin
GFP-actinhttp://www.einstein.yu.edu/aif/instructions/tirf/index.htm
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Superresolution microscopy
Beyond the diffraction limit
d = 0.61 λ / NA
Imaging EGFP in living cells has a resolution of approximately 200 (XY) and 500 nanometers (Z)
Confocal
STEDSample courtesy Martin Engelke, Urs Greber, Institute of Zoology, University of Zurich
Super resolutionmicroscopy
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Super resolutionmicroscopy
Enhanced PSF microscopy Statistical microscopy
STEDStimulated emission depletion
STORMStochastic optical reconstruction microscopy
PALMPhotoactivated localization microscopy
SSIMSaturated structured illumination microscopy
GSDGround state depletion microscopy
Stimulated emission depletion microscopy
STED
In STED, an initial excitation pulse is focused on a spot.
The spot is narrowed by a second, donut-shaped pulse that prompts all excited fluorophores in the body of the donut to emit (this is the “emission depletion” part of STED).
This leaves only the hole of the donut in an excited state, and only this narrow hole is detected as an emitted fluorescence.
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Saturated structured illumination microscopy
SSIM
Sample structure Illumination pattern Image (Moiré)
Algorithm (calculation of sample structure)
Statistical microscopy –Imaging single molecules
GSDGround state depletion microscopy
PALMPhotoactivated localization microscopy
STORMStochastic optical reconstruction microscopy
Position of a single molecule can be localized to 1 nmaccuracy or better if enough photons are collected andthere are no other similarly emitting molecules within~200 nm (Heisenberg 1930, Bobroff 1980).
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Statistical microscopy –Imaging single molecules
GSDGround state depletion microscopy
PALMPhotoactivated localization microscopy
STORMStochastic optical reconstruction microscopy
Position of a single molecule can be localized to 1 nmaccuracy or better if enough photons are collected andthere are no other similarly emitting molecules within~200 nm (Heisenberg 1930, Bobroff 1980).
Price and DavidsonFlorida State University
Thank you Literature
Fundamentals of light microscopy and electronic imaging, Douglas B. Murphy; Wiley-Liss, 2001ISBN 0-471-25391-X
Light Microscopy in Biology – A practical approach,A. J. Lacey; Oxford University Press, 2004
Light and Electron Microscopy, E. M. Slayter, H. S. Slayter; Cambridge University Press, 1992
http://microscopy.fsu.edu/primer/index.html